The detection of biological molecules and the quantification of reaction processes is a central tool of biochemical research. A long-felt need exists for improved methods for the detection of molecular species of biological interest. Such a need exists in particular for the detection of soluble peptidoglycans or of the antibiotics which react with soluble peptidoglycans or with antibodies to soluble peptidoglycans in liquids, especially in bodily fluids or in soil samples, or of antibodies which have specificity towards soluble peptidoglycans.
Peptidoglycans in general have been known to have a variety of biological properties, including the ability to activate the complement system and to stimulate macrophages and lymphocytes. In addition, they are under consideration as possible factors in the origin of some diseases of unknown cause such as systemic rheumatic diseases. Soluble forms of peptidoglycans (SPG) are believed to be murein B lymphocyte activators. A correlation has also been drawn between SPG and certain forms of endocarditis; they may plan a role in other mammalian diseases as well. A sensitive, reliable and specific method for the detection of SPG in biological and other fluids is essential for further medical and biochemical studies. Moreover, general methods for the detection of biological and other molecules are also needed.
Enzyme linked immunosorbent assay (ELISA) has been known heretofore for the detection of antigens. Prior ELISA techniques have bound an antibody from a first species to a surface, which antibody is selectively bindable to the antigen to be detected. The surface-bonded antibody is then contacted by a liquid suspected to contain the antigen. The antigen binds to the surface-bonded antibody, thus, in turn, being bound to the surface. An antibody from a second species, also specific for the antigen to be detected, is then introduced and allowed to bind to the bound antigen. The resulting combination can be referred to as a "sandwich" comprising surface, first antibody, antigen, and second antibody.
An enzymatic system is then attached to or associated with the second antibody in the "sandwich" through a number of techniques. The most common is the use of an enzymatically-labelled antibody from a third species with specificity for the antibody (immunoglobulin) from the second species. The enzymatic system is then provided with substrate and allowed to operate to produce products. The diminution of enzyme substrate or the increase in enzyme products or both is monitored and related to the number of "sandwiches", and hence the number of antigen molecules, which have been bound to the surface.
The ELISA technique suffers from a lack of flexibility. Thus, in order to perform prior ELISA techniques, it is necessary to identify, prepare and isolate two antibodies for each antigen to be detected and to ensure that the two antibodies can bind the antigen simultaneously in the "sandwich". There has been a long-felt need for immunosorbent assays which do not require plural antibodies and which are capable of assaying additional, detectable species with improved efficiency and precision.
Two antibodies may have similarities in their binding sites and thus may bind the same analogues of the natural material to be detected, thereby lowering the specificity and ensuring high backgrounds in the ELISA. Unlike antibodies, antibiotics are not elicited by the natural material to be detected and have a much different binding site for the material. Thus, analogues of the material to be detected that bind to the antibody may well not bind to the antibiotic. Furthermore, plural antibodies from different species may still have some antigenic sites in common. If so, the antibody from the third species may have some specificity to the antibody from the first species, thereby lowering the specificity and ensuring high backgrounds in the ELISA. The antibody from the third species against antibody (immunoglobulin) from the second species is extremely unlikely to cross-react with an antibiotic.